The transmission of disease by blood products and other biological materials remains a serious health problem. While significant advances in blood donor screening and blood testing have occurred, viruses such as hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus (HIV) may escape detection in blood products during testing due to low levels of virus or viral antibodies. In addition to the viral hazard, there are currently no adequate licensed tests to screen for the presence of non-viral microbes, such as bacteria or protozoans, in blood intended for use in transfusions. The risk also exists that a hitherto unknown pathogen may become prevalent in the blood supply and present a threat of disease transmission, as in fact occurred before the recognition of the risk of HIV transmission via blood transfusions.
Chemical agents have been introduced into blood or blood plasma to inactivate pathogens prior to clinical use of the blood product. Typically, for blood products having little or no red blood cell content, such as platelets and plasma, photochemically activated compounds such as psoralens are used. For red blood cell-containing blood products, compounds have been developed for pathogen inactivation, which do not require photoactivation. These compounds typically have electrophilic groups that react with pathogens, more specifically with pathogen nucleic acid. For example, U.S. Pat. No. 5,055,485 describes the inactivation of viruses in cell and protein-containing compositions using aryl diol epoxides. Other compounds that generate electrophiles in situ may be used. LoGrippo et al. evaluated the use of nitrogen mustard, CH3—N(CH2CH2Cl)2, for viral inactivation. LoGrippo et al., Proceedings of the Sixth Congress of the International Society of Blood Transfusion, Bibliotheca Haematologica (Hollander, ed.), 1958, pp. 225-230. More significantly, U.S. Pat. Nos. 5,691,132; 6,177,441; 6,410,219; 6,143,490; and 6,093,725, the disclosures of which are hereby incorporated by reference, describe the use of compounds that have a nucleic acid targeting component as well as an electrophilic component that reacts with the nucleic acid in order to inactivate the pathogen. U.S. Pat. Nos. 6,093,725 and 6,514,987, the disclosures of which are hereby incorporated by reference, describe similar compounds, wherein the nucleic acid targeting component of the compound is linked to the reactive electrophilic component via a hydrolysable linker. U.S. Pat. Nos. 6,136,586 and 6,617,157, the disclosures of which are hereby incorporated by reference, describe using ethyleneimine oligomers and related compounds for pathogen inactivation. These ethyleneimine-derived compounds typically have an aziridine group, which provides the reactive electrophilic component, and a polyamine component, which provides nucleic acid targeting of the compound. The general class of nucleic acid targeted compounds having an electrophilic or similar group reactive with the nucleic acid are used to inactivate pathogens in blood, blood products, and a variety of samples of biological origin.
There is some concern that, while these compounds are designed to specifically target nucleic acids, they may react with other components of the blood, such as proteins or cellular membranes. These side-reactions are unfavorable and may cause adverse effects, such as modifications of proteins and cell membranes that may be recognized by the immune system. When such treated blood products are used repeatedly, they may result in an immune response of the recipient to the treated blood product. U.S. Pat. Nos. 6,270,952; 6,709,810; and 7,293,985, the disclosures of which are hereby incorporated by reference, describe methods of quenching such pathogen-inactivating compounds in order to reduce the level of any such adverse side-reactions. U.S. Patent Publication No. 2006/0115466, the disclosure of which is hereby incorporated by reference, describes improvements to these quenching conditions which address an immune response developed against the pathogen-inactivating compound. However, despite the improvement in quenching effectiveness, in some cases the treated red blood cells have been found to have decreased lifespan attributed to increased cell dehydration as measured by decreased osmotic fragility and decreased spun hematocrit.
Thus, there is a need for methods to reduce unwanted electrophilic side-reactions of pathogen-inactivating compounds while preserving the ability of the pathogen-inactivating compound to inactivate harmful pathogens without adversely affecting the vitality and lifespan of the treated blood product. Specifically, there is a need for improved methods of quenching pathogen-inactivating compounds in red blood cells.